Typical known examples of cell-penetrating peptides (CPPs) include TAT peptides, penetratin peptides, and artificially synthesized peptides comprising 7 to 9 arginine residues. It is known that these peptides are rich in arginine, easily recognize negatively charged substances on the cell surface, and enter cells by endocytotic mechanism.
Such cell-penetrating peptides have been used for intracellular delivery of substances that are difficult to deliver intracellularly, including large molecular substances such as proteins or nucleic acids, and even small molecules. However, such conventional peptides require micromolar concentrations to exhibit a sufficient ability to penetrate cells. These cell-penetrating peptides need to be covalently conjugated to the substance to be delivered into cells in order to increase the cell-penetrating ability of the substance. Thus, these peptides have insufficient efficiency. This is because physiologically active substances (such as proteins or small molecules) to be delivered into cells mostly perform their function at concentrations lower than micromolar concentrations. Thus, efforts have been made to develop a technology that enables cell-penetrating peptides to penetrate cells with high efficiency at lower concentrations.
In recent years, efforts have been made to prepare a stapled peptide from a portion of a physiologically active protein in order to regulate the physiological activity thereof. Such stapled peptides for regulating physiological activity mostly consist of hydrophobic amino acids. This is because amino acids playing an important role in protein-protein interactions consist mainly of hydrophobic functional groups.
Recently, the inventors of the application prepared a peptide multimer comprising covalent linkages at two or more amino acid positions of amphipathic alpha-helical peptides comprising hydrophilic and hydrophobic amino acids (PCT/KR2014/009778). It was found that this peptide multimer can be used as a cell-penetrating peptide. Particularly, as described in PCT/KR2014/009778, the present inventors previously prepared dimeric peptides by introducing cysteine instead of leucine into some hydrophobic residues of amphipathic peptides and connecting the peptides by two disulfide bonds. It was found that such dimeric peptides have a greatly increased alpha-helical content, are chemically stable, and have a cell-penetrating ability which is about 500-fold higher than conventional CPPs.
However, if one peptide of a dimeric peptide has 16 amino acid residues, the dimeric peptide consists of 32 amino acids. Thus, in order to develop a better cell-penetrating peptide, it is required to prepare a stapled peptide and compare the cell-penetrating ability thereof with that of a dimeric peptide.
Under this technical background, the present inventors have found that, based on a stapled peptide, a cell-penetrating peptide that is a peptide monomer can be prepared which has an improved cell-penetrating ability while maintaining its chemical stability, thereby completing the present invention.